This invention relates to sterilization indicators for use in monitoring the effectiveness of sterilization procedures that use liquid sterilants, and in particular to sterilization indicators for use in monitoring the effectiveness of sterilization procedures that use liquid peracetic acid as a sterilant.
In hospitals, industry and research it is necessary to sterilize certain articles, such as glassware, medical instruments and the like, to ensure that they are completely free of contamination by microorganisms that are capable of causing infection and disease. For example, it is well known that surgical instruments must be sterilized before they may be used in a surgical operation. Articles are sterilized by placing them in a machine called a sterilizer and subjecting them to a sterilization procedure of a type that is generally recognized as being effective at killing microorganisms.
Many different types of sterilization procedures are known in the art. These procedures may be differentiated from each other based on the identity of the sterilant used in each to destroy contaminating microorganisms. Currently, the most widely used procedures employ steam or an antimicrobial gas as sterilants.
However, sterilization procedures have recently been developed that use liquid sterilants. In particular, sterilization procedures that use liquid peracetic acid as a sterilant have become increasingly popular of late because they may be used to sterilize articles that cannot withstand the high temperatures and pressures of steam sterilization. A liquid peracetic acid sterilization procedure is described in U.S. Pat. No. 5,077,008 (Kralovic).
Sterilization indicators are quality control devices that are included with every load of articles placed in a sterilizer to monitor the antimicrobial effectiveness of each procedure conducted by the sterilizer. The indicators determine whether or not a sterilization procedure was lethal to contaminating microorganisms in the load. Typically, in the event of sterilization failure the indicators provide a readily detectable signalxe2x80x94such as fluorescence, luminescence or a color changexe2x80x94indicating that contaminating microorganisms may have survived and that the articles in the load must be subjected to another sterilization cycle.
Sterilization indicators have been developed that are both rapid and accurate for monitoring steam and gas procedures. But until now, a critical shortcoming associated with the use of liquid sterilant procedures has been that sterilization indicators have not been available that meet the performance standards set for sterilization indicators used with steam or gas procedures. In general, sterilization indicators for use with liquid sterilants are neither as fast or as dependable as sterilization indicators developed for use with steam.
Effective sterilization indicators that are widely used to monitor steam and gas sterilization procedures include biological indicators, rapid readout biological indicators, and dual rapid readout biological indicators. With each of these types of sterilization indicators, the failure of a sterilization procedure is indicated by a detectable change in a biological specimenxe2x80x94either an enzyme, a microorganism or bothxe2x80x94that is attached to a carrier substrate, such as filter paper. In theory all of these sterilization indicators should be just as effective with liquid sterilants as they are with steam or gas. However, in practice it is often the case that the biological specimen is washed off of the carrier material during the procedure, or the carrier material is degraded by the liquid sterilant, so that any results obtained are unreliable.
Biological indicators have heretofore been recognized in the art as providing an accurate and direct method for determining whether or not a sterilization procedure has been effective, for steam and gas. Biological indicators are sterilization indicators that monitor the effectiveness of a sterilization procedure by measuring the effect of the procedure on a population of test microorganisms included in the indicator. If a sterilization procedure fails to generate a condition in the sterilizer that is lethal to the test microorganisms, surviving cells are detectable following a period of incubation and growth.
The test microorganism used in a biological indicator is typically a microorganism that is several times more resistant to the sterilization procedure being monitored than any microorganism that would likely to be present due to natural contamination. Spores of a microorganism, such as a bacterium, fungus, or protozoan, are most commonly used as the test microorganisms in biological indicators because of their hardy resistance to sterilization procedures. Bacillus stearothermophilus spores are preferred for use in biological indicators for monitoring steam sterilization procedures, while Bacillus subtilis spores are preferred for use in biological indicators for ethylene oxide gas procedures.
Many biological indicators currently in use are self-contained biological indicators, which include both a spore strip and growth medium in separate compartments within the same closed container. During a sterilization procedure, sterilant, either steam or gas, enters the container through a shielded vent and contacts the spores but does not contact the growth medium. The barrier between the separate compartments is then broken, and the growth medium and spores are combined and incubated. Typically, a pH indicator compound that changes color in response to spore outgrowth is included with the growth medium. If test spores survive the sterilization procedure and grow out during incubation, the growth medium changes color, providing a readily detectable signal that the sterilization procedure has failed. A major advantage of self-contained biological indicators is that they can be sterilized, incubated and read without ever opening the container and exposing its contents to potential contamination from microorganisms present in the environmental air.
A typical self-contained biological indicator is described in U.S. Pat. No. 3,661,717 (Nelson). The indicator includes a compressible outer tube having one open end, and a porous, protective material covering the open end that allows sterilant, but not bacteria, to enter and leave the tube. A sealed glass ampoule within the outer tube contains growth medium and a chemical compound that changes color in response to spore growth. The outer tube also contains a spore strip between the outer tube and the ampoule. During a sterilization procedure steam or gas sterilant enters the indicator through the cover material and contacts the spores on the spore strip. The glass ampoule is then crushed by squeezing the compressible walls of the outer tube, and the spores are combined with the growth media. The indicator is incubated for a prescribed period of time and observed for a color change indicating spore growth and sterilization failure.
Attempts have been made in the art to develop biological indicators that are suitable for use with liquid sterilants. U.S. Pat. No. 4,885,253 (Kralovic) discloses a biological indicator for use with liquid sterilants that includes a vial with a sealed compartment containing growth media and a vented cap that is capable of being moved from an open position in which sterilant may freely enter the vial to a closed position. A spore strip is disposed above the sealed compartment and is exposed to sterilant during the sterilization procedure. Afterwards, a cutter device attached to the inner surface of the cap severs the barrier separating the growth media from the rest of the compartment, causing the spore strip to drop into the growth media for incubation.
U.S. Pat. No. 5,736,355 (Dyke) discloses a biological indicator for use with liquid sterilants that includes a vial with a sealed compartment containing growth medium and a vented cap that moves from an open position in which sterilant can freely enter the container to a closed position. A dart with an attached spore strip is attached to the cap for puncturing the seal and immersing the spore strip in the culture medium for incubation. The spore strip is covered by a microporous membrane that is said to prevent the spores from being washed away by liquid sterilant during a sterilization procedure.
U.S. Pat. No. 5,516,648 (Malchesky) discloses a biological indicator for use with liquid sterilants that includes a spore strip encapsulated in a microporous, hydrophylic membrane that is permeable to sterilant but impermeable to spores. The encapsulated spore strip is located above a sealed media compartment in a vial, and is exposed to sterilant during a sterilization procedure. It is said that the microporous membrane prevents spores from being washed off the spore strip by sterilant. The vial includes a cap with a cutting device for rupturing the seal after a sterilization procedure in order to drop the encapsulated spore strip into the growth media.
Although biological indicators are accurate, they are slow and may require up to seven days to give a final result. Ideally, articles that have been exposed to a sterilization procedure should be quarantined until biological indicator results have confirmed that the procedure was effective. However, as a practical matter this is rarely done. Limitations on storage space and the constant need for freshly sterilized supplies make it difficult, if not impossible, to hold goods in reserve inventory for the long period of time necessary for biological indicators to provide a result. Instead, ostensibly sterilized goods are often put to immediate use, and later-obtained negative biological indicator results are used to identify and remedy existing sterility problems, rather than to prevent them from occurring in the first instance.
Rapid readout biological indicators have been developed that avoid these problems by combining the accuracy of biological indicators with the speed necessary for medicine and industry. These indicators rely on an enzyme-substrate reaction that provides a detectable indication of sterilization accuracy within hours or minutes, rather than days as required with conventional biological indicators.
Rapid readout biological indicators are described in U.S. Pat. No. 5,073,488 (Matner et al.), U.S. Pat. No. 5,418,167 (Matner et al.), and U.S. Pat. No. 5,223,401 (Foltz et al.), which are incorporated herein by reference. These indicators have a self-contained design that includes an enzyme and a substrate that is capable of reacting with the enzyme, in separate compartments within the same container. In a preferred design, the indicators include a compressible outer tube with one open end and a porous, protective cover membrane over the open end that is permeable to sterilant but impermeable to bacteria. A sealed glass ampoule within the outer container contains a substrate solution. A carrier strip made of filter paper or some other appropriate material is located between the walls of the outer tube and the ampoule, and includes on its surface a source of an active enzyme having an enzyme activity that is correlated with the survival of a test microorganism that is commonly used in biological indicators to monitor sterilization procedures. During a sterilization procedure, sterilant enters the outer tube and contacts the enzyme located the carrier strip. The inner ampoule is then crushed, and the enzyme and substrate combined and incubated. If any enzyme on the carrier remains active after the sterilization procedure, it will react with the substrate to form an enzyme-modified product that is detectable as luminescence, fluorescence or a color change indicating sterilization failure.
Dual rapid readout biological indicators are rapid readout indicators that measure the effectiveness of a sterilization procedure with both a rapid enzyme-substrate reaction and a confirmatory spore outgrowth test of the type used in conventional biological indicators. 3M ATTEST(trademark) 1292 Rapid Readout Indicators, available from 3M Company, St. Paul, Minn., for use in monitoring steam sterilization procedures, are dual rapid readout indicators in which Bacillus stearothermophilus spores serve as both the source of active enzyme for the enzyme test and the test microorganism for the confirmatory outgrowth test. The spores are coated on the surface of a carrier strip that is located between the walls of a compressible outer tube and a sealed glass ampoule containing growth medium and substrate. After sterilization the glass ampoule is crushed by squeezing the outer tube, and the spores and the contents of the ampoule are combined and incubated. The indicator is examined for fluorescence, indicating sterilization failure, after 3 hours using a 3M ATTEST(trademark) Model 190 Rapid Auto reader, and then is incubated for a full 7 days for the confirmatory spore outgrowth test.
Although rapid readout sterilization indicators and dual rapid readout sterilization indicators are highly accurate, it has not previously been possible to use them with liquid sterilants, because of the tendency of the sterilant to wash the source of enzyme from the indicators. It is therefore an object of the present invention to provide rapid readout biological indicators and dual rapid readout indicators that overcome this problem and that may be used to accurately and reliably monitor a sterilization procedure that employs a liquid sterilant.
The invention provides a rapid readout biological indicator that may reliably be used to monitor sterilization procedures that disinfect articles with liquid sterilants, including liquid peracetic acid. The indicator relies upon an enzyme-substrate reaction to provide a rapid indication of whether or not a sterilization procedure has been effective. The invention overcomes the known tendency of liquid sterilants to wash biological test specimens out of biological indicators, rendering them unreliable, by providing the biological test specimen of the indicator in a protective gel matrix coated on a surface within the indicator housing. Sterilant may enter the matrix and contact the biological test material, but the biological material remains enmeshed in the matrix and is not washed away by the sterilant.
The indicator of the invention includes a compressible outer container having one open end that is shielded by a cover membrane that is permeable to liquids but impermeable to bacteria. An enzyme-gel matrix is coated on a surface within the outer container. The enzyme-gel matrix includes a source of active enzyme dispersed within a water-insoluble, biologically inert gel. A breakable sealed ampoule within the outer container contains a substrate that is capable of reacting with the active enzyme in the enzyme-gel matrix. During a sterilization procedure, sterilant enters the outer tube through the cover membrane and contacts the source of active enzyme in the enzyme-gel matrix. The substrate ampoule is then broken by compressing the outer tube, and the substrate and enzyme-gel matrix are combined and incubated. If the sterilization procedure is ineffective, active enzyme will react with substrate to form a product that provides a detectable indication that the sterilization procedure has failed and must be repeated.
The enzyme-gel matrix has physical properties such that during a sterilization procedure, sterilant may enter the matrix and contact the source of active enzyme without washing the source of active enzyme out of the indicator. The enzyme used in the indicator has an enzyme activity that is correlated with the survival of at least one test microorganism that is commonly used to monitor the effectiveness of a sterilization procedure. The enzyme is substantially inactivated by a sterilization procedure that is lethal to the test microorganism, but is not substantially inactivated by a sterilization procedure that is not lethal to the test microorganism.
The source of active enzyme used in the indicator of the invention may be a purified enzyme, a microorganism or both. If a microorganism is used as the source of active enzyme, the indicator may be a dual rapid readout biological indicator in which the microorganism serves as both the source of active enzyme for the rapid enzyme test and the microorganism for the outgrowth test. A growth medium is included in the substrate ampoule in the dual rapid readout biological indicator to facilitate growth of any surviving spores after exposure to a sterilization procedure.
In an embodiment of the indicator that is suitable for monitoring liquid peracetic acid sterilization procedures, the source of active enzyme is Bacillus stearothermophilus spores.
The enzyme-gel matrix in the indicator of the invention may be coated on any surface within the outer tube that is exposed to sterilant during a sterilization procedure. For example, it may be coated on the interior surface of the outer tube, on the exterior surfaces of the substrate ampoule, or on a carrier material such as filter paper or a glass bead.